The present invention relates to a composition and to a method of imparting resistivity to superinfection with HIV.
More in particular the invention concerns a composition and a method to confer resistance to human cells, in particular T-cells, to the superinfection with a retrovirus, specifically the human immunodeficiency virus (HIV).
Several experimental strategies aimed at blocking HIV replication in vivo have been proposed. So far, most have been based on immunotherapeutic or chemotherapeutic approaches, in which the anti-HIV mechanism of action is well known. However, none of the immunotherapeutic and chemotherapeutic approaches proposed thus far has been demonstrated to effect a resolutive anti-HIV therapy.
The goal of a more recently developed alternative anti-HIV experimental design was to render the target cell resistant to HIV replication through the induction of intracellular immunization (Baltimore, Nature 335: 395-396 (1988)). Inhibition of the superinfecting HIV was, in fact, demonstrated in cells expressing HIV-trans-dominant proteins (Malim et al., J. Exp. Med. 176: 1197-1201 (1992); Green et al., Cell 58:215-223 (1989); Modesti et al., New Biol. 3:759-768 (1991); Trono et al., Cell 59:113-120 (1989), Lisziewicz et al., Annual Meeting, Laboratory of Tumor Cell Biology, Gene Therapy (1993); Buchschacher et al., Hum. Gene Ther. 3:391-397 (1992); Stevenson et al., Cell 83:483-486 (1989); and Liu et al., Gene Therapy 1:32-37 (1994)), HIV genome-directed ribozymes (Yu et al., Gene Therapy 1:13-26 (1994); Lorentzen et al., Virus Genes 5:17-23 (1991); Sioud et al., PNAS USA 88:7303-7307 (1991); Weerasinghe et al., J. Virol. 65:5531-5534 (1991); and Yamada et al., Gene Therapy 1:38-45 (1994)), tat/rev decoys (Sullenger et al., Cell 63:601608 (1990); Sullenger et al., J. Virol. 65:6811-6816 (1991); and Smith et al., UCLA/UCI AIDS Symposium: Gene Therapy Approaches to Treatment of HIV Infection (1993)), or antisense RNA (Rhodes et al., J. Gen. Virol. 71:1965-1974 (1990); Rhodes et al., AIDS 5: 145-151 (1991), Sczakiel et al., J. Virol. 65:468-472 (1991); Joshi et al., J Virol. 65:5524-5530 (1991), and Chatterjee et al., Science 258:1485-1488 (1992)). Moreover, an effective anti-HIV intracellular immunization was achieved by transfecting HIV-susceptible cells with DNA coding for either an anti-gpl60 single chain antibody (Marasco et al., PNAS USA 90:7889-7893 (1993)) or a monoclonal anti-rev single chain variable region (Duan et al., Abstract from the 1994 Annual Meeting, Laboratory of Tumor Cell Biology, Sep. 25-Oct. 1, 1994, MD USA).
The prior art methods, however, suffer from the following disadvantages. Many of the above mentioned methods do not work properly in practice because of the fact that anti-HIV compounds able to target a single step of the HIV replication could easily induce the emergence of HIV resistant mutants. This is a very common biological event, considering the extraordinary ability of HIV to mutate as demonstrated, for example, by the HIV strains resistant to antiretroviral chemical compounds (i.e., AZT, ddI) isolated from AIDS patients treated with such drugs. Consequently, many investigators are attempting to synthesize anti-HIV reagents able to target different steps of the HIV life cycle. In addition, some of the described methods have been demonstrated to be either deleterious for the host cells, or difficult to be effectively applicable in clinical protocols.
The present invention seeks to overcome the disadvantages of the above-described methods. The authors of the invention have set up a method to confer resistitivity to human cells, in particular to T-cells, to HIV superinfection, following to a stable integration of an HIV non-infective non-producer genomic variant into at least some of cell nuclear genomes. In order to confer resistivity to human cells to HIV superinfection, the instant invention provides a composition to be used for HIV intracellular immunization, during the AIDS therapy. the invention has several advantages, including an easy way of administration, the lacking of HIV spontaneous reversion of non defective variants from a non producer to a producer phenotype, and the ability to confer a good resistance to superinfection.
Evidence obtained by in vitro experiments indicates that F12-HIV non defective non producer variant expression could inhibit different steps of the wild type HIV superinfecting life cycle. In fact, the authors of the invention have shown a block of the wild-type HIV replication, eithr before or after its own tetrotrascrption, depending on the F12-HIV expressing cells tested. Moreover, no modifications or impairments of the physiologic cell functions were demonstrated in any cells harbouring the F12-HIV genome.
xe2x80x9cHIVxe2x80x9d is used herein to encompass all designations past and present assigned to those viruses implicated as causative agents of acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC), such as HIV, e.g., HIV-1 and HIV-2, and HTLV, e.g., HTLV-III. By xe2x80x9csuperinfectionxe2x80x9d is meant that which is known to and understood by those of ordinary skill in the art, i.e., the ability of a given virus to infect a cell already infected by a virus. By xe2x80x9cresistivityxe2x80x9d is meant the capacity to resist superinfection by a virus, in particular a retrovirus, specifically HIV. By xe2x80x9cnondefectivexe2x80x9d is meant that the genome is complete, whereas by xe2x80x9cnonproducerxe2x80x9d is meant that viral particles are not produced.
The instant invention providees a composition comprising a retroviral vector comprising a non-defective, non-producer, HIV variant genome, in a sufficient amount to get a stable integration of said non-defective, non-producer, HIV variant genome, in at least some of nuclear genomes of target cells, and the expression of said non-defective, non-producer, HIV variant genome in said target cells, in order to confer resistivity to HIV superinfection to cells, in particular to T cells.
Preferably, the non-defective, non-producer, HIV variant genome integrates in at least the 1% of cell nuclear genomes. More preferably the non-defective, non-producer, HIV variant genome integrates in from about 1% to about 10% of cell nuclear genomes. Preferably the non-defective, non-producer, HIV variant genome is the HIV F12 genome. It is further preferred that the 3xe2x80x2 LTR of the F12-HIV genome be deleted prior to insertion into the retroviral vector and that the genome be inserted into the retroviral vector in antisense orientation. A retroviral vector derived from Moloney murine leukemia virus is preferred for the composition of the invention.
A further aspect of the invention provides the use of a retroviral vector comprising a non-defective, non-producer, HIV variant genome in sufficient amount to get a stable integration of the non-defective, non-producer, HIV variant genome into at least some of the nuclear genomes of the cells get in contact with said vector and the expression into said cells of said nondefective, nonproducer HIV variant genome.
Preferably, the non-defective, non-producer, HIV variant genome integrates in at least the 1% of cell nuclear genomes. More preferably the non-defective, non-producer, HIV variant genome integrates in from about 1% to about 10% of cell nuclear genomes. Preferably the non-defective, non-producer, HIV variant genome is the HIV F12 genome. It is preferred that the 3xe2x80x2 LTR of the F12-HIV genome be deleted prior to insertion into the retroviral vector and that the genome be inserted into the retroviral vector in antisense orientation. A retroviral vector derived from Moloney murine leukemia virus is preferred for the composition of the invention.
Alternatively the instant invention provides the use of a composition comprising a retroviral vector a non-defective, non-producer, HIV variant genome to be utilised for the production of a AIDS therapy medicament.
An applicative method of the invention is of imparting resistivity to HIV superinfection to human cells, by means of removing cells from an human, contacting the removed-cells with the composition of the invention in sufficient amount to get a stable integration and the expression of the nondefective, nonproducer HIV variant into at least some of the nuclear genomes of the cells, and reintroducing the so-treated cells to the human-from which the cells had been removed. Such ex vivo methods are described in Ferrari G., Rossini S., Giavazzi R., Maggioni D., Nobili N., Soldati M., Ungers G., Mavilio F., Gilboa E., Bordignon C. xe2x80x9cAn in vivo model of somatic cell gene therapy for human severe combined immunodeficiency.xe2x80x9d Science 251: 1363, 1991.
Although, theoretically, any genome from a nondefective, nonproducer HIV variant could be useful or rendered useful in the present inventive method, the genome from the F12-HIV (Dr. M. Federico, Laboratory of Virology, Istituto Superiore di Sanita, Rome).
The F12-HIV variant genome, whose sequence and charcterization is described in Carlini et al. J. Viral Disease 1:40-55 (1992) here wholly incorporated, is characterized by more than 50 mutations, most of which are in the gag, pol , and vif genes and which do not appear to be present in other HIV-1 strains. The mechanism of resistance to superinfection is unknown, and it is conceivable that the resistance is caused either by the expression of a single negative trans-dominant gene product or, more likely, by the concomitant expression of more than one such a product. The exchange of homologous fragments between the F12-HIV variant genome and a wild-type, infectious HIV-1 molecular clone, named pNL4-3 (Dr. M. Martin, AIDS Research and Reference Program, AIDS Program, NIAID, NIH), followed by transfection into HIV-sensitive human cells, indicates that it is necessary to replace at least 6 kb of the genome, between the Bcl I and Xho I restriction sites and encompassing the pol, vif, vpr, vpu, tat, rev, and env genes in order to revert the F12 phenotype from nonproducer to producer. Accordingly, the risk of spontaneous reversion of the nondefective F12-HIV variant from replication-deficient to replication-competent is appreciably low. This risk is further reduced by the absence of the entire 3xe2x80x2 LTR and most of the nef gene, both of which are necessary for replication, in the preferred F12-HIV variant constructs. A cell population homogeneously expressing the F12 HIV genome would block any contaminant replication-competent HIV, by the interfering action of the same F12-HIV; this is consistent with evidence that, in HIV-1 superinfected HeLa CD4+ cells expressing the full-length F12-HIV provirus, no infectious HIV-1 release was observed in over three months of continuous CEMss co-culture.
The inactivation of the F12 genes, alone and in all possible combinations, either by mutagenesis, e.g., insertion of a premature stop codon, or by deletion, followed by transfection of the resultant mutants into HIV-permissive human cells can be performed to determine which mutation (s) enable(s) the F12 genome to confer resistance to superinfection. Alternatively, every single mutation in the F12 genome can be reverted to wild-type and the resulting products transfected into HIV-permissive human cells. Such methods would enable the identification of the gene(s) responsible for the interference mechanism and only those genes could be mutated in other HIV genomes, thereby enabling the use of other HIV genomes in the context of the present inventive method.
Although, theoretically, any retroviral vector is suitable or can be rendered suitable for use in the present inventive method, a retroviral vector that allows expression of the F12 genome at sufficiently high levels to effect viral interference, i.e., resistance to superinfectdon, should be used. In addition, the retroviral vector should express a selective gene, preferably the gene encoding the resistance to the G418 antibiotic. Examples of retroviral vectors include N2, pLj, LXSN, and NSV. The retroviral vector that is preferred for use in the present inventive method is N2. Vectors derived from the Moloney murine leukemia virus (MoMLV) (Dr. E. Gilboa, Sloan-Kettering Institute for Cancer Research, New York, N.Y.) are especially preferred.
Measures should be taken to avoid any instability caused by the presence of two identical, repeated sequences, i.e., the 5xe2x80x2 and 3xe2x80x2 LTRs, in the vector. A preferred method involves deletion of the 3xe2x80x2 LTR, which contains part of the nef gene, of the F12-HIV genome prior to insertion in the retroviral vector.
Measures should also be taken to avoid any transcriptional interference, such as that which can result between the LTRs of F12 and of the retroviral vector. A preferred measure involves insertion of the F12 genome, in particular the F12 genome from which the 3xe2x80x2 LTR has been deleted, into the retroviral vector in antisense orientation.
In accordance with the present invention, the composition comprising a retroviral vector comprising a genome from a nondefective, nonproducer HIV variant as described above is administered directly to a human. Suitable ways of administration are known to those of ordinary skill in the art. Irrespective of which composition and method of administration are used, the recombinant retroviral vector should be administered in sufficient amount to the human to effect stable integration and expression of the nondefective, nonproducer HIV variant genome into at least some of the nuclear genomes of the cells of the human (see, e.g., Ferrari et al., Science 25: 1363 (1991); Ferrari et al., Blood 80: 1120 (1992), and Mavilio et al., Blood 83: 1988 (1994) and the protocol set forth in Borneo et al., Human Gene Therapy 4: 513 (1993)).
The retroviral vector comprising the nondefective, nonproducer HIV variant genome can be made into a composition appropriate for contacting cells in vitro, or pharmaceutical compositions appropriate for administration in vivo, with appropriate carriers or diluents, which further can be pharmaceutically acceptable. Where appropriate, the vectors can be formulated into preparations in solid, semisolid, liquid or gaseous forms, such as suppositories, injections, inhalants, and aerosols, in the usual ways for their respective way of administration. Means known in the art can be utilised to prevent release and absorption of the composition until it reaches the desired cells or to ensure timed-release of the composition. A pharmaceutically acceptable form should be employed which does not ineffectuate the composition. In pharmaceutical dosage forms, the compositions can be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
The pharmaceutical compositions of the present invention can be delivered through different ways and to different sites in the body. One skilled in the art will recognise that, although more than one way can be used for administration, a particular way can provide a more immediate and more effective result than another way. Local or systemic delivery can be accomplished by administration comprising application or instillation of the composition into body cavities, inhalation or insufflation of an aerosol, or by parental introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.
The compositions of the present invention can be provided in unit dosage form, wherein each dosage unit, e.g., a teaspoonful, tablet, solution, or suppository, contains a predetermined amount of the composition, alone or in appropriate combination with other active agents. The term xe2x80x9cunit dosage formxe2x80x9d as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the composition of the present invention, alone or in combination with other active agents, calculated-in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate. The specifications for unit dosage forms depend on the particular pharmacodynamics of the particular individual to be treated.
Accordingly, a recombinant retroviral vector comprising a nondefective, nonproducer HIV-1 variant genome can be administered to a human using any of the aforementioned ways of administration or alternative ways known to those of skill in the art and appropriate for a particular application. The amount of the vector administered should be sufficient to effect stable integration of the nondefective, nonproducer HIV variant genome into at least some of the nuclear genomes of the cells in the human and expression of the nondefective, nonproducer HIV variant genome therein. Such integration can be monitored, for example, by evidence of genome integration, e.g., using the polymerase chain reaction in conjunction with sequencing or Southern hybridizations.
The present invention composition can be used to effect intracellular immunization so as to prevent or, at the very least, substantially inhibit, initial HIV infection in an individual at risk for such an infection. It can also be used in the therapeutic treatment of an HIV+ individual by blocking or, at the very least, slowing the spread of the infection and preventing or, at the very least, delaying the onset of AIDS or ARC. In this regard, the composition can be used in combination with other antiretroviral drugs, in particular other anti-HIV treatments, provided that they do not adversely affect the ability of the nondefective, nonproducer HIV variant genome to stably integrate into at least some of the nuclear genomes of the cells in the human and to express itself therein. It should be noted that the present composition also has utility in vitro. For example, the composition could be used to study, besides further aspects concerning F12 HIV induced interference (i.e., in the SCID mouse in vivo model or in HIV chronically infected cells), the effects of HIV encoded proteins on the physiology of ex vivo cells (i.e., peripheral blood lymphocytes, monocytes, astrocytes) in the absence of viral replication.